Ischemic cardiomyopathy (ICM) is the most prevalent type of chronic heart diseases in the United States and a common underlying etiology of congestive heart failure. Nonetheless the underlying molecular mechanism of ICM remains unclear. Moreover, the prognosis of chronic ICM is very poor if left untreated. Therefore it is highly important to gain a better understanding of the disease mechanism and to identify specific biomarkers for early detection and treatment of ICM. Myofilament proteins in the sarcomeres not only play essential roles in cardiac contractility but also are critical elements in signal reception and transduction during the onset and progression to heart failure. The hypothesis is that post-myocardial infarction (MI) maladaptive cardiac remodeling can result in altered protein modifications in myofilaments that are associated with cardiac dysfunction and offer potential diagnostic and prognostic biomarkers of ICM. Although multiple myofilament post-translational modifications (PTMs) are believed to act in concert in regulating cardiac function, a comprehensive analysis of myofilament proteins simultaneously and assess all synergistic PTM changes related to cardiac function is lacking. Herein, we will employ an innovative integrated top-down protein mass spectrometry (MS)-based disease proteomics platform to simultaneously examine myofilament proteins extracted from healthy and diseased swine and human myocardium and identify all disease-related changes in myofilament modifications. Aim 1 will identify altered protein modifications in myofilaments from diseased swine myocardium of post-MI ICM model and determine their functional consequences. Aim 2 will determine myofilament alterations in end-stage failing human myocardium from ICM patients and their functional consequences. Aim 3 will assess the functional effects of novel myofilament modifications in Ca2+-mediated cardiac muscle contraction and relaxation. Mechanical properties (the isometric force and the Ca2+-sensitivity of the force) will be measured in skinned myocardial preparations in parallel to proteomics experiments. Furthermore, we will assess regional systolic and diastolic function to establish mechanistic links between specific proteomic changes and in vivo and ex vivo cardiac function. With the completion of this project, we expect to identify multiple functionally significant alterations in myofilaments and understand how these alterations act in concert to modulate maladaptive signaling during post-MI left ventricular remodeling to failure. The success of our research will provide new insights into the mechanisms underlying ICM and will identify new candidate biomarkers for early detection of the presence and progression of ICM.